Protein phosphatase-1 catalytic subunit interactions

Abstract

A method of identifying a compound which modulates the interaction between a PP1c and a regulatory subunit thereof, the method comprising determining whether a compound enhances or disrupts the interaction between (a) a PP1c or a fragment, variant, derivative or fusion thereof or a fusion of a fragment, variant or derivative and (b) a regulatory subunit which is able to bind to PP1c or a PP1c-binding fragment, variant, derivative or fusion of the subunit or a fusion of the fragment, variant or derivative. A method of affecting cellular metabolism or function, the method comprising administering to a cell (a) a compound which modulates the interaction between a PP1c and a regulatory subunit thereof or (b) a compound which mimics the effect of a regulatory subunit of PP1c or (c) a peptide capable of binding a PP1c and which affects the ability of PP1c to bind to a particular target and/or affects the regulation of PP1c activity, or a functional equivalent thereof.

1. A method of identifying a compound which modulates the interaction between a PP1c and a regulatory subunit thereof, the method comprising assaying for the interaction between (a) a PP1c and (b) a regulatory subunit which is able to bind to PP1c or a PP1c-binding fragment or variant, wherein the fragment or variant comprises SEQ ID NO:35, or fusion of said subunit or a fusion of said fragment or variant, wherein the regulatory subunit is any one of M 110 , G L , G M , M-complexes, p53 BP2, sds22, NIPPI, L5, Inhibitor-1, Inhibitor-2, or DARPP, and assaying for the interaction in the presence of a compound, to identify a compound which modulates the interaction. 
     
     
       2. A method of identifying a compound which mimics the effect of a regulatory subunit of PP1c, the method comprising assaying for the enzymatic or binding activity of PP1c in the presence of a given regulatory subunit, and contacting a compound with PP1c and determining whether, in the presence of the compound, PP1c adopts the enzymatic or binding activity of the PP1c in the presence of the given regulatory subunit, wherein if PP1c adopts the enzymatic or binding activity of the PP1c in the presence of a given regulatory subunit, the compound mimics the effect of the regulatory subunit of PP1c, and wherein the regulatory subunit is any one of M 110 , G L , G M , M-complexes, p53 BP2, sds22, NIPPI, L5, Inhibitor-1, Inhibitor-2, or DARPP. 
     
     
       3. A method according to  claim 1  or  2  wherein the regulatory subunit of PP1c is any one of M 110 , G L , G M , M-complexes or p53BP2. 
     
     
       4. A method according to  claim 3  wherein the regulatory subunit of PP1c is M 110  or G M . 
     
     
       5. A method according to  claim 1  wherein the fragment of a regulatory subunit which is able to bind to PP1c is any of the peptides G63-T93 of SEQ ID NO:32, G63-N75 of SEQ ID NO:32, E2-P243 of SEQ ID NO:34, E2-D118 of SEQ ID NO:34, and peptide 63-80 of SEQ ID NO:32 G M  or peptides comprising said peptide sequences provided that they are not the complete G M  regulatory subunit. 
     
     
       6. A method according to  claim 1  wherein the fragment of a regulatory subunit which is able to bind to PP1c is any of the peptides M1-E309 of SEQ ID NO:33, M1-F38 of SEQ ID NO:33, M1-A150 of SEQ ID NO:33, or L24-Y496 of SEQ ID NO:33 of M 110  or peptides comprising said peptide sequences provided that they are not the complete M 110  regulatory subunit. 
     
     
       7. A method according to  claim 1  wherein the PP1c-binding fragment or variant further comprises at least one basic residue in the four residues N-terminal of the consensus peptide sequence. 
     
     
       8. A method according to  claim 1 , wherein the third amimo acid in SEQ ID NO:35 is not Asp or Glu or a large hydrophobic residue. 
     
     
       9. A method according to  claim 1  wherein the PP1c-binding fragment is a fragment of a regulatory subunit. 
     
     
       10. A method according to  claim 8  wherein the PP1c-binding fragment is a fragment of any of the M 110 , G L , G M , M-complexes, p53BP2, sds22, NIPPI, L5, Inhibitor-1, Inhibitor-2 or DARPP regulatory subunits comprising said SEQ ID NO:35. 
     
     
       11. A method according to  claim 1  or  2  wherein the compound binds to a PP1c. 
     
     
       12. A method according to  claim 1  wherein the compound binds to a regulatory subunit of PP1c. 
     
     
       13. A compound which modulates the interaction between a PP1c and a regulatory subunit thereof said compound comprising any of the peptides G63-T93 of SEQ ID NO:32, G63-N75 of SEQ ID NO:32, E2-P243 of SEQ ID NO:34, E2-D118 of SEQ ID NO:34, and peptide 63-80 of SEQ ID NO:32 G M  or said compound comprising any of the peptides M1-E309 of SEQ ID NO:33, M1-F38 of SEQ ID NO:33, M1-A150 of SEQ ID NO:33 or L24-Y496 of SEQ ID NO:33 of M 110  or said compound comprising the consensus peptide sequence SEQ ID NO:35:Arg/Lys-Val/Ile-Xaa-Phe wherein Xaa is any naturally occurring amino acid, provided that said compound is not a complete regulatory subunit of PP1c. 
     
     
       14. A compound according to  claim 13  consisting of the peptides G63-T93 of SEQ ID NO:32, G63-N75 of SEQ ID NO:32, E2-P243 of SEQ ID NO:34, E2-D118 of SEQ ID NO:34, or peptide 63-80 of SEQ ID NO:32 G M  or consisting of the peptides M1-E309 of SEQ ID NO:33, M1-F38 of SEQ ID NO:33, M1-A150 of SEQ ID NO:33 or L24-Y496 of SEQ ID NO:33 of M 110 . 
     
     
       15. A method of identifying a compound which modulates the interaction between a PP1c and a regulatory subunit thereof, or binds PP1c or mimics the effect of a regulatory subunit, the method comprising determining the conformation of a peptide bound to a regulatory subunit-binding site of PP1c and the conformation of the portion of PP1c which binds to the peptide, and selecting a compound which is capable of adopting the same conformation as the peptide bound to the regulatory subunit-binding site of PP1c or the same conformation as the portion of PP1c which binds to said peptide, wherein the selected compound modulates the interaction between a PP1c and a regulatory subunit thereof or binds PP1c and a regulatory subunit thereof or binds PP1c or mimics the effect of a regulatory subunit, wherein the regulatory subunit is any one of M 110 , G L , G M , M-complexes, p53BP2, sds22, NIPPI, L5, Inhibitor-1, Inhibitor-2 or DARPP. 
     
     
       16. A method according to  claim 15  wherein said peptide comprises the consensus peptide sequence SEQ ID NO:35: Arg/Lys-Val/Ile-Xaa-Phe wherein Xaa is any amino acid. 
     
     
       17. A method according to  claim 16  wherein said peptide consists of residues 63 to 75 of SEQ ID NO: 32 G m . 
     
     
       18. A composition comprising a compound according to  claim 13  and an acceptable carrier. 
     
     
       19. A method according to  claim 2  wherein the function or properties of the PP1c include the subcellular location of the PP1c, the substrate specificity of the PP1c, the activity of the PP1c towards one or more substrates, the activity of the PP1c in the presence of one or more extracellular agonists, the ability of the PP1c to bind to a regulatory subunit, or the ability of the PP1c to be modulated by reversible protein phosphorylation or second messengers.